Two-step hybridization and capture of a polynucleotide

Abstract

A method for capturing a target polynucleotide in a sample onto a solid support with an attached immobilized probe by using a capture probe and two different hybridization conditions, which preferably differ in temperature only, is disclosed. The two hybridization conditions control the order of hybridization, where the first hybridization conditions allow hybridization of the capture probe to the target polynucleotide, and the second hybridization conditions allow hybridization of the capture probe to the immobilized probe. The method may be used to detect the presence of a target polynucleotide in a sample by detecting the captured target polynucleotide or amplified target polynucleotide.

1 . A method of capturing a target polynucleotide present in a sample, comprising the steps of: 
 a) incubating a mixture comprising a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe:target hybridization complex comprising the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe:capture probe hybridization complex comprising the immobilized probe and the capture probe; and    b) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe:capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe:capture probe:target hybridization complex comprising the immobilized probe, the capture probe and the target polynucleotide.    
     
     
         2 . The method of  claim 1 , wherein the first incubating step uses a temperature below a T m  of the capture probe:target hybridization complex and above a T m  of the immobilization probe:capture probe hybridization complex, and wherein the second incubating step uses a temperature below a T m  of the immobilization probe:capture probe hybridization complex.  
     
     
         3 . The method of  claim 2 , wherein the second incubating step comprises lowering the temperature of the first hybridization condition by at least about 10° C.  
     
     
         4 . The method of  claim 2 , wherein the second incubating step comprises lowering the temperature of the first hybridization condition by at least about 20° C.  
     
     
         5 . The method of  claim 2 , wherein the first incubating step uses a temperature of about 60° C. and the second incubating step uses a temperature of about 40° C. or lower.  
     
     
         6 . The method of  claim 1 , wherein the first incubating step uses a solution having a chemical stringency that favors formation of the capture probe:target hybridization complex and disfavors formation of the immobilization probe:capture probe hybridization complex, and wherein the second incubating step lowers the chemical stringency of the solution thereby favoring formation of the immobilization probe:capture probe hybridization complex.  
     
     
         7 . The method of  claim 1 , further comprising the step of purifying the immobilized probe:capture probe:target hybridization complex.  
     
     
         8 . The method of  claim 7 , further comprising the step of detecting the target polynucleotide in the purified immobilized probe:capture probe:target hybridization complex.  
     
     
         9 . The method of  claim 8 , wherein the detecting step comprises hybridizing a labeled probe to the target polynucleotide in the purified immobilized probe:capture probe:target hybridization complex.  
     
     
         10 . The method of  claim 9 , wherein the detecting step further comprises removing the labeled probe that has not hybridized to the target polynucleotide.  
     
     
         11 . The method of  claim 1 , further comprising the step of detecting the target polynucleotide in the immobilized probe:capture probe:target hybridization complex.  
     
     
         12 . The method of  claim 11 , wherein the detecting step comprises hybridizing a labeled probe to the target polynucleotide.  
     
     
         13 . The method of  claim 12 , wherein the detecting step further comprises removing the labeled probe that has not hybridized to the target polynucleotide.  
     
     
         14 . The method of  claim 1 , further comprising the step of amplifying the target polynucleotide to produce an amplified nucleic acid.  
     
     
         15 . The method of  claim 14 , wherein the amplifying step comprises transcription-associated amplification.  
     
     
         16 . The method of  claim 15 , further comprising the step of detecting the amplified nucleic acid.  
     
     
         17 . The method of  claim 16 , wherein the detecting step comprises hybridizing a labeled probe to the amplified nucleic acid that has a sequence complementary to the target polynucleotide sequence.  
     
     
         18 . The method of  claim 17 , wherein the detecting step further comprises removing the labeled probe that has not hybridized to the amplified nucleic acid.  
     
     
         19 . The method of  claim 1 , wherein: 
 the immobilized probe comprises a capture probe-binding region of at least five nucleotide base recognition groups in length, and    the capture probe comprises an immobilized probe-binding region of at least five nucleotide base recognition groups in length,    and wherein the capture probe-binding region is complementary to the immobilized probe-binding region.    
     
     
         20 . The method of  claim 19 , wherein the capture probe-binding region of the immobilized probe comprises: 
 (a) a first backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and    (b) at least ten nucleotide base recognition groups joined to the first backbone, wherein each nucleotide base recognition group is capable of hydrogen bonding with adenine, guanine, cytosine, thymine, uracil or inosine;    and wherein the immobilized probe-binding region of the capture probe comprises:    (a) a second backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and    (b) at least ten nucleotide base recognition groups joined to the second backbone, which are capable of hydrogen bonding to the nucleotide base recognition groups joined to the first backbone.    
     
     
         21 . The method of  claim 19 , wherein the capture probe-binding region of the immobilized probe consists of a repetitious base sequence of at least 10 nucleotide base recognition groups, and the immobilized probe-binding region of the capture probe consists of a repetitious base sequence comprising at least 25 nucleotide base recognition groups, of which at least 10 nucleotide base recognition groups are complementary to the capture probe-binding region.  
     
     
         22 . The method of  claim 21 , wherein the capture probe-binding region comprises a sequence of about fourteen contiguous A or T, and the immobilized probe-binding region comprises a sequence of 30 bases complementary thereto.  
     
     
         23 . The method of  claim 1 , wherein the capture probe and the immobilized probe each comprise deoxynucleotide, ribonucleotide, 2′-methoxy substituted nucleotide, 2′-halo substituted nucleotide components, or combinations thereof.  
     
     
         24 . A method for determining the presence of a target polynucleotide in a sample, comprising the steps of: 
 a. providing a capture probe capable of hybridizing to a target polynucleotide present in a sample;    b. mixing the capture probe with a sample suspected of containing the target polynucleotide at a first incubation temperature that favors hybridization of the capture probe and the target polynucleotide, thereby producing a capture probe:target polynucleotide complex;    c. providing an immobilized probe capable of hybridizing to the capture probe;    d. incubating a mixture comprising the capture probe:target polynucleotide complex and the immobilized probe at a second incubation temperature that favors hybridization of the immobilized probe and the capture probe, thereby producing a captured target polynucleotide comprising an immobilized probe:capture probe:target polynucleotide complex;    e. purifying the captured target polynucleotide, thereby producing a purified target polynucleotide;    f. amplifying the purified target polynucleotide, thereby producing an amplified nucleic acid; and    g. detecting the amplified nucleic acid which indicates the presence of the target polynucleotide in the sample.    
     
     
         25 . The method of  claim 24 , wherein the detecting step detects an amplified nucleic acid that is complementary to the target polynucleotide or a portion thereof.  
     
     
         26 . The method of  claim 25 , wherein the detecting step comprises using a labeled probe to detect the amplified nucleic acid.